The broad, long term objectives are to utilize the cytoprotective abilities of heparin-binding EGF-like growth factor (HB-EGF) in the treatment of intestinal ischemia/reperfusion (I/R) injury. The goal of the present study is to determine the means by which HB-EGF decreases expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS) within the intestine following I/R injury. We have shown that administration of HB-EGF during the ischemic phase of an I/R episode in rats substantially attenuates the extent of intestinal tissue damage and decreases mortality; as well, the post-I/R expression of iNOS, systemic appearance of nitric oxide, and generation of ROS within the affected intestine are significantly decreased. Using in vitro studies, we have demonstrated that treatment with HB-EGF decreases cytokine- induced upregulation of iNOS in human intestinal epithelial (DLD- 1) cells, and reduces generation of ROS by leukocytes and rat intestinal epithelial cells. Our hypothesis is that HB-EGF decreases tissue injury following I/R by limiting the increased expression of iNOS and ROS that occur in this setting. To test this hypothesis, the application has two specific aims: (I) To delineate the protective effects of HB-EGF in an in vivo model of intestinal I/R injury, and (II) To elucidate the mechanisms by which HB-EGF attenuates the post-I/R increase in iNOS and ROS. The research design and methods are to clarify issues regarding the dose, route, and timing of HB-EGF administration in vivo, and to use our animal model of intestinal I/R to characterize the effects of HB-EGF on expression of iNOS, ROS, antioxidant enzymes and pro-inflammatory cytokines by the intestine post-I/R. In Aim II, DLD-1 cells will be used to determine if HB-EGF alters the rate of iNOS mRNA transcription or its stability, and if NF- kappaB activation occurs following HB-EGF administration. Human umbilical vein endothelial cells will be used to determine if HB- EGF affects the conversion of xanthine dehydrogenase to the oxidase form, and human leukocytes will be used to determine the effects of the peptide on NAPDH oxidase expression and function. The health relatedness of this work is to eventually perform clinical trials of the use of HB-EGF in patients with intestinal I/R injury, and the data generated in this proposal will allow us to obtain important information regarding the mechanisms of HB- EGF intestinal cytoprotection.